The ability to polarize is a fundamental property of living cells, whether they are epithelial, nerve, or migrating cells, or simply growing cells that have to pick an axis for cell division. To begin to understand the structural basis for cell polarity, we employ the budding yeast because exquisite molecular and classical genetics, biochemistry and cell biology are available in this organism. Moreover, the basic principles that yeast uses to polarize its secretory pathway, and probably how it segregates its organelles during cell division, are very similar to the mechanisms employed by vertebrate cells. In the current period, we have shown that yeast has a special organizing center that drives the assembly of tropomyosin-containing actin cables. These cables are the mechanical substrates for polarized movement by the unconventional myosin-V encoded by yeast MYO2. We have shown that Myo2p binds through its tail to secretory vesicles and transports them down the actin cables for polarized growth. We have also found that the initial alignment of the nucleus with the axis of cell division is achieved through the association of the Myo2p cargo domain with Kar9p to deliver it into the bud. We propose three specific aims that build on these studies. First, we propose to identify components involved in assembling and regulating the polarized actin cables. We focus on the yeast formins, Bnilp and Bnrlp, as these appear to be key scaffolding proteins involved in the process of cable formation. Through genetic analysis we propose approaches to identify components involved in actin cable assembly, and then, based on these studies, propose approaches to reconstitute this process in vitro. Second, we propose to examine what other organdlies might be segregated by Myo2p, and approaches to identify molecules that allow Myo2p to transport specific cargoes. Third, since Myo2p is the best understood member of the myosin-V superfamily, we propose to analyze the structure of its cargo binding domain by both dissecting specific activities genetically as well as determining its structure at the atomic level. These studies will provide a foundation for understanding how yeast sets up a polarized cytoskeleton and in turn uses it to target polarized growth and segregate organelles during the cell cycle. Since homologues of molecules critical for these processes in yeast have been associated with defects in mice and in human diseases, this work should be of general relevance.